Epigallocatechin-3-gallate Protects Against Cisplatin-induced Nephrotoxicity By Inhibiting Endoplasmic Reticulum Stress-induced Apoptosis

Background and ObjectiveCisplatin (CP) is an effective chemotherapeutic agent that is widely accepted as a treatment for various types of malignancies. However, its serious adverse effects, especially nephrotoxicity, restrict its clinical use. CP induces renal tubular dysfunction and leads to acute renal failure in a large proportion of patients. The pathogenesis of CP-induced nephrotoxicity can be attributed to many factors, including oxidative stress, inflammation, necrosis, apoptosis and ischemia. Renal tubular apoptosis plays an important role in CP-induced nephrotoxicity. Several pathways have been studied in CP-induced apoptosis, including the extrinsic pathway, the endoplasmic reticulum (ER) stress pathway and the intrinsic mitochondrial pathway. ER stress pathway has been implicated to play an important role in CP-induced apoptosis. Epigallocatechin-3-gallate (EGCG), the major ingredient of green tea, has potent anti-apoptotic, antioxidant, and anti-inflammatory properties, which can be used for preventing many diseases. However, the protective effect of EGCG on CP-induced nephrotoxicity still remains unknown. Herein, we used EGCG to interfere with the process of CP-induced nephrotoxicity in many different ways, including measuring renal function, observing the pathologic changes in the kidney, examining the number of nephritic apoptosis and testing the expressions of caspase-12 and glucose regulating protein78 (GRP78). The present study was designed to investigate the protective effects of EGCG against CP-induced nephrotoxicity and explore its renal protective mechanism.MethodsTwenty-eight adult male C57/BL6 mice were randomly divided in the following groups:(1) Control group (N; n=7) were treated with 0.9% saline (lOml/kg, ip); (2) EGCG group (E; n=7) were treated with EGCG (100mg/kg, ip) in 0.9% saline (10mg/ml); (3) CP group (C; n=7) were treated with CP (20.0 mg/kg, ip) in 0.9% saline (2mg/ml); (4) CP+EGCG group (C+E; n=7) were pretreated with EGCG (100mg/kg, ip) at 30 min before ip injection of CP (20.0 mg/kg). After 48h of CP injection, mice in this group were treated with EGCG (100mg/kg, ip).5 days (120 hours) after injection of CP or 0.9% saline, all animals were weighed and sacrificed. Kidney tissues and blood were collected after sacrificing. The serum was separated from blood by centrifugation (3000 rpm,15 min, at 4℃) and stored at -80℃. Serum levels of blood urea nitrogen (BUN) and creatinine (Scr) were measured using Cobas(?)8000 modular analyzer (Roche Diagnostics). We immediately weighed the kidneys and calculated the renal index (RI) as both kindeys’ weight (g)/animal’s weight (g) x100. We used periodic acid-Schiff (PAS) staining to examine the pathologic changes in the kidney, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) to examine the number of nephritic apoptosis, and used western blotting and immunohistochemical staining to examine the protein levels of caspase-12 and GRP78.Results(1) Renal function and renal index changes:Compared to the control group, the levels of BUN, Scr, and RI increased dramatically in the CP group as shown in Table 1 (P<0.05), which were significantly decreased by EGCG treatment in the CP+EGCG group (P<0.05). (Table 1)(2) Renal pathological changes:In the CP group, renal proximal tubule cells revealed vacuolation, necrosis, and desquamation, while in the control and EGCG groups, the proximal tubule cells maintained normal morphology as shown in Figure la. CP treatment resulted in severe tubular damage, which was attenuated by EGCG treatment in the CP+EGCG group (P<0.05). (Figure 1)(3)TUNEL results:TUNEL-positive apoptotic cell number raised significantly in the CP group compared to the control group (P<0.05), and were reduced in the CP+EGCG group (P<0.05). TUNEL-positive cells in the control and the EGCG group were very limited as shown in Figure 2.(4)The expression of GRP78 and caspase-12:The results showed that CP injection significantly increased the expression of GRP78 and caspase-12 in the CP group compared to the control group (P<0.05). However, the expression of these proteins was significantly attenuated with EGCG treatment in the CP+EGCG group (P<0.05). (Figures 3 and 4)ConclusionEGCG can protect against CP-induced nephrotoxicity by suppression of ER stress-mediated apoptosis.